Purpose.

AMD results in loss of central vision and a dependence on low-resolution peripheral vision. While many image enhancement techniques have been proposed, there is a lack of quantitative comparison of the effectiveness of enhancement. We developed a natural visual search task that uses patients' eye movements as a quantitative and functional measure of the efficacy of image modification.

Methods.

Eye movements of 17 patients (mean age = 77 years) with AMD were recorded while they searched for target objects in natural images. Eight different image modification methods were implemented and included manipulations of local image or edge contrast, color, and crowding. In a subsequent task, patients ranked their preference of the image modifications.

Results.

Within individual participants, there was no significant difference in search duration or accuracy across eight different image manipulations. When data were collapsed across all image modifications, a multivariate model identified six significant predictors for normalized search duration including scotoma size and acuity, as well as interactions among scotoma size, age, acuity, and contrast (P < 0.05). Additionally, an analysis of image statistics showed no correlation with search performance across all image modifications. Rank ordering of enhancement methods based on participants' preference revealed a trend that participants preferred the least modified images (P < 0.05).

Conclusions.

There was no quantitative effect of image modification on search performance. A better understanding of low- and high-level components of visual search in natural scenes is necessary to improve future attempts at image enhancement for low vision patients. Different search tasks may require alternative image modifications to improve patient functioning and performance.

We examined the effect of image modification on visual search performance in patients with central vision loss from AMD. We found no significant effect on performance measures across eight different image modifications.

Rationale

Despite its incorporation into research studies, the safety aspects of segmental allergen bronchoprovocation and differences in cellular response among different allergens have received limited consideration.

Methods

We performed 87 segmental challenges in 77 allergic asthma subjects. Allergen dose was based on each subject’s response to whole lung allergen challenge. Bronchoalveolar lavage was performed at 0 and 48 hours. Safety indicators included spirometry, oxygen saturation, heart rate, and symptoms.

Results

Among subjects challenged with ragweed, cat dander, or house dust mite, there were no differences in safety indicators. Subjects demonstrated a modest oxygen desaturation and tachycardia during the procedure that returned to normal prior to discharge. We observed a modest reduction in forced vital capacity and forced expiratory volume in one second following bronchoscopy. The most common symptoms following the procedure were cough, sore throat and fatigue. Total bronchoalveolar lavage fluid cell numbers increased from 13±4 to 106±108×104 per milliliter and eosinophils increased from 1±2 to 44±20 percent, with no significant differences among the three allergens.

Conclusions

In mild allergic asthma, segmental allergen bronchoprovocation, using individualized doses of aeroallergens, was safe and yielded similar cellular responses.

New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis (M. tb) are urgently needed. We report on the identification of an adamantyl urea compound displaying potent bactericidal activity against M. tb and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm where they are synthesized to the periplasmic side of the plasma membrane where they are transferred onto cell wall arabinogalactan or used in the formation of virulence-associated outer membrane trehalose-containing glycolipids. Whole genome sequencing of spontaneous resistant mutants of M. tb selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter, MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane.

The treatment of tuberculosis is becoming more difficult due to the ever increasing prevalence of drug resistance. Thus, it is imperative that novel anti-tuberculosis agents, with unique mechanisms of action, be discovered and developed. The direct anti-tubercular testing of a small compound library led to discovery of adamantyl urea hit compound 1. In this study, the hit was followed up through the synthesis of an optimization library. This library was generated by systematically replacing each section of the molecule with a similar moiety until a clear structure activity relationship was obtained with respect to anti-tubercular activity. The best compounds in this series contained a 1-adamantyl-3-phenyl urea core and had potent activity against Mycobacterium tuberculosis plus an acceptable therapeutic index. It was noted that the compounds identified and the pharmacophore developed is consistent with inhibitors of epoxide hydrolase family of enzymes. Consequently, the compounds were tested for inhibition of representative epoxide hydrolases: M. tuberculosis EphB and EphE; and human soluble epoxide hydrolase. Many of the optimized inhibitors showed both potent EphB and EphE inhibition suggesting the antitubercular activity is through inhibition of multiple epoxide hydrolyase enzymes. The inhibitors also showed potent inhibition of humans soluble expoxide hydrolyase, but limited cytotoxicity suggesting that future studies must be towards increasing the selectivity of epoxide hydrolyase inhibition towards the M. tuberculosis enzymes.

Introduction

This phase I/II study evaluated the safety and anti-tumor effect of the combination of erlotinib with cixutumumab, a recombinant fully humanized anti-insulin-like growth factor-1 receptor IgG1 monoclonal antibody, in advanced non-small cell lung cancer (NSCLC).

Methods

Patients with advanced NSCLC were treated in an initial safety-lead and drop-down cohorts using erlotinib 150 mg/d with cixutumumab 6 or 5 mg/kg on days 1, 8, 15, and 22 in 28-day cycles (cohorts 1 and 2). Emerging pharmacokinetic data led to an additional cohort (3 + 3 design) with cixutumumab at 15 mg/kg on day 1 in 21-day cycles (cohort 3).

Results

Eighteen patients entered the study (6 at 6 mg/kg, 8 at 5 mg/kg, and 4 at 15 mg/kg), with median age of 65 years. Four of six patients at 6 mg/kg experienced dose-limiting toxicities (DLTs), whereas at 5 mg/kg, one of eight patients experienced DLT but three of eight patients still required a dose delay during cycle 1. At 15 mg/kg every 21 days, two of four patients experienced DLTs. In all cohorts, DLTs were either G3 rash or fatigue. Five patients had stable disease as best response and 14 patients had progressive disease. The median progression-free survival was 39 days (range 21–432+ days). Biomarkers analyses showed a trend toward better progression-free survival seen with higher free baseline insulin-like growth factor-1 levels as seen with other insulin-like growth factor-1R inhibitors.

Conclusions

The combinations of cixutumumab at 6 mg/kg every 7 days and 15 mg/kg every 21 days and full-dose erlotinib are not tolerable in unselected patients with NSCLC, as measured by DLT. Cixutumumab at 5 mg/kg every 7 days was tolerable per DLT, but dose delays were common. Efficacy in unselected patients with NSCLC seems to be low.

We report the synthesis and biochemical evaluation of several selective inhibitors of class II (zinc dependent) fructose bis-phosphate aldolases (Fba). The products were designed as transition-state analogues of the catalyzed reaction, structurally related to the substrate fructose bis-phosphate (or sedoheptulose bis-phosphate) and based on an N-substituted hydroxamic acid, as a chelator of the zinc ion present in active site. The compounds synthesized were tested on class II Fbas from various pathogenic microorganisms and, by comparison, on a mammalian class I Fba. The best inhibitor shows Ki against class II Fbas from various pathogens in the nM range, with very high selectivity (up to 105). Structural analyses of inhibitors in complex with aldolases rationalize and corroborate the enzymatic kinetics results. These inhibitors represent lead compounds for the preparation of new synthetic antibiotics, notably for tuberculosis prophylaxis.

Background

Chronic pulmonary disease and skin/soft tissue infections due to non-tuberculous mycobacteria (NTM) of the Mycobacterium chelonae-abscessus-massiliense group is an emerging health problem worldwide. Moreover, the cure rate for the infections this group causes is low despite aggressive treatment. Post-surgical outbreaks that reached epidemic proportions in Brazil recently were caused by M. massiliense isolates resistant to high-level disinfection with glutaraldehyde (GTA). Understanding the differences in the virulence and host immune responses induced by NTM differing in their sensitivity to disinfectants, and therefore their relative threat of causing outbreaks in hospitals, is an important issue.

Methodology/Principal Finding

We compared the replication and survival inside macrophages of a GTA-susceptible reference Mycobacterium massiliense clinical isolate CIP 108297 and an epidemic strain from Brazil, CRM-0019, and characterized the immune responses of IFNγ knockout mice exposed to a high dose aerosol with these two isolates. CRM-0019 replicated more efficiently than CIP 108297 inside mouse bone marrow macrophages. Moreover, the animals infected with CRM-0019 showed a progressive lung infection characterized by a delayed influx of CD4+ and CD8+ T cells, culminating in extensive lung consolidation and demonstrated increased numbers of pulmonary CD4+ Foxp3+ regulatory T cells compared to those infected with the reference strain. Immunosuppressive activity of regulatory T cells may contribute to the progression and worsening of NTM disease by preventing the induction of specific protective immune responses.

Conclusions/Significance

These results provide the first direct evidence of the increased virulence in macrophages and mice and pathogenicity in vivo of the Brazilian epidemic isolate and the first observation that NTM infections can be associated with variable levels of regulatory T cells which may impact on their virulence and ability to persist in the host.

Arabinosyltransferases are a family of membrane-bound glycosyltransferases involved in the biosynthesis of the arabinan segment of two key glycoconjugates, arabinogalactan and lipoarabinomannan, in the mycobacterial cell wall. All arabinosyl-transferases identified have been found to be essential for the growth of Mycobcterium tuberculosis and are potential targets for developing new antituberculosis drugs. Technical bottlenecks in designing enzyme assays for screening for inhibitors of these enzymes are (1) the enzymes are membrane proteins and refractory to isolation; and (2) the sole arabinose donor, decaprenylphosphoryl-d-arabinofuranose is sparingly produced and difficult to isolate, and commercial substrates are not available. In this study, we have synthesized several analogues of decaprenylphosphoryl-d-arabinofuranose by varying the chain length and investigated their arabinofuranose (Araf) donating capacity. In parallel, an essential arabinosyltransferase (AftC), an enzyme that introduces α-(1→3) branch points in the internal arabinan domain in both arabinogalactan and lipoarabinomannan synthesis, has been expressed, solubilized, and purified for the first time. More importantly, it has been shown that the AftC is active only when reconstituted in a proteoliposome using mycobacterial phospholipids and has a preference for diacylated phosphatidylinositoldimannoside (Ac2PIM2), a major cell wall associated glycolipid. α-(1→3) branched arabinans were generated when AftC–liposome complex was used in assays with the (Z,Z)-farnesylphosphoryl d-arabinose linear α-d-Araf-(1→5)3–5 oligosaccharide acceptors and not with the acceptor that had a α-(1→3) branch point preintroduced.

No chemoprevention strategies have been proven effective for lung cancer.

We evaluated the effect of 13-cis retinoic acid (13-cis RA), with or without α tocopherol, as a lung cancer chemoprevention agent in a phase II randomized controlled clinical trial of adult subjects at high risk for lung cancer as defined by the presence of sputum atypia, history of smoking, and airflow obstruction, or a prior surgically cured nonsmall cell lung cancer (disease free, >3 years). Subjects were randomly assigned to receive either 13-cis RA, 13-cis RA plus α tocopherol (13-cis RA/α toco) or observation for 12 months.

Outcome measures are derived from histologic evaluation of bronchial biopsy specimens obtained by bronchoscopy at baseline and follow-up. The primary outcome measure is treatment “failure” defined as histologic progression (any increase in the maximum histologic score) or failure to return for follow-up bronchoscopy.

Seventy-five subjects were randomized (27/22/26 to obervations/13-cis RA/13-cis RA/α toco); 59 completed the trial; 55 had both baseline and follow-up bronchoscopy. The risk of treatment failure was 55.6% (15 of 27) and 50% (24 of 48) in the observation and combined (13 cis RA plus 13 cis RA/α toco) treatment arms, respectively (odds ratio adjusted for baseline histology, 0.97; 95% confidence interval, 0.36–2.66; P = 0.95). Among subjects with complete histology data, maximum histology score in the observation arm increased by 0.37 units and by 0.03 units in the treated arms (difference adjusted for baseline, −0.18; 95% confidence interval, −1.16 to 0.81; P = 0.72). Similar (nonsignificant) results were observed for treatment effects on endobronchial proliferation as assessed by Ki-67 immunolabeling.

Twelve-month treatment with 13-cis RA produced nonsignificant changes in bronchial histology, consistent with results in other trials. Agents advancing to phase III randomized trials should produce greater histologic changes. The addition of α tocopherol did not affect toxicity.

Purpose

Management of empyema has been debated in the literature for decades. Although both primary video-assisted thoracoscopic surgery (VATS) and tube thoracostomy with pleural instillation of fibrinolytics have been shown to result in early resolution when compared to tube thoracostomy alone, there is a lack of comparative data between these modes of management. Therefore, we conducted a prospective, randomized trial comparing VATS to fibrinolytic therapy in children with empyema.

Methods

After Institutional Review Board approval, children defined as having empyema by either loculation on imaging or more than 10,000 white blood cells/μL were treated with VATS or fibrinolysis. Based on our retrospective data using length of postoperative hospitalization as the primary end point, a sample size of 36 patients was calculated for an α of .5 and a power of 0.8. Fibrinolysis consisted of inserting a 12F chest tube followed by infusion of 4 mg tissue plasminogen activator mixed with 40 mL of normal saline at the time of tube placement followed by 2 subsequent doses 24 hours apart.

Results

At diagnosis, there were no differences between groups in age, weight, degree of oxygen support, white blood cell count, or days of symptoms. The outcome data showed no difference in days of hospitalization after intervention, days of oxygen requirement, days until afebrile, or analgesic requirements. Video-assisted thoracoscopic surgery was associated with significantly higher charges. Three patients (16.6%) in the fibrinolysis group subsequently required VATS for definitive therapy. Two patients in the VATS group required ventilator support after therapy, one of whom required temporary dialysis. No patient in the fibrinolysis group clinically worsened after initiation of therapy.

Conclusions

There are no therapeutic or recovery advantages between VATS and fibrinolysis for the treatment of empyema; however, VATS resulted in significantly greater charges. Fibrinolysis may pose less risk of acute clinical deterioration and should be the first-line therapy for children with empyema.

The reemergence of tuberculosis in its present-day manifectations – single, multiple and extensive drug resistant forms and as HIV-TB coinfections – has resulted in renewed research on fundamental questions such as the nature of the organism itself, Mycobacterium tuberculosis, the molecular basis of its pathogenesis, definition of the immunological response in animal models and humans, and development of new intervention strategies such as vaccines and drugs. Foremost among these developments has been the precise chemical definition of the complex and distinctive cell wall of M. tuberculosis, elucidation of the relevant pathways and underlying genetics responsible for the synthesis of the hallmark moities of the tubercle bacillus such as the mycolic acid-arabinogalactan-peptidoglycan complex, the phthiocerol- and trehalose-containing effector lipids, the phosphatidylinositol-containing mannosides, lipomannosides and lipoarabinomannosides, major immunomodulators, and others. In this review, the laboratory personnel that have been the focal point of some to these developments review recent progress towards a comprehensive understanding of the basic physiology and functions of the cell wall of M. tuberculosis.

Arabinogalactan (AG) and lipoarabinomannan (LAM) are the two major cell wall (lipo)polysaccharides of mycobacteria. They share arabinan chains made of linear segments of α-1,5-linked d-Araf residues with some α-1,3-branching, the biosynthesis of which offers opportunities for new chemotherapeutics. In search of the missing arabinofuranosyltransferases (AraTs) responsible for the formation of the arabinan domains of AG and LAM in Mycobacterium tuberculosis, we identified Rv0236c (AftD) as a putative membrane-associated polyprenyl-dependent glycosyltransferase. AftD is 1400 amino acid-long, making it the largest predicted glycosyltransferase of its class in the M. tuberculosis genome. Assays using cell-free extracts from recombinant Mycobacterium smegmatis and Corynebacterium glutamicum strains expressing different levels of aftD indicated that this gene encodes a functional AraT with α-1,3-branching activity on linear α-1,5-linked neoglycolipid acceptors in vitro. The disruption of aftD in M. smegmatis resulted in cell death and a decrease in its activity caused defects in cell division, reduced growth, alteration of colonial morphology, and accumulation of trehalose dimycolates in the cell envelope. Overexpression of aftD in M. smegmatis, in contrast, induced the accumulation of two arabinosylated compounds with carbohydrate backbones reminiscent of that of LAM and a degree of arabinosylation dependent on aftD expression levels. Altogether, our results thus indicate that AftD is an essential AraT involved in the synthesis of the arabinan domain of major mycobacterial cell envelope (lipo)polysaccharides.

p-Hydroxybenzoic acid derivatives (p-HBADs) are glycoconjugates secreted by all Mycobacterium tuberculosis isolates whose contribution to pathogenicity remains to be determined. The pathogenicity of three transposon mutants of M. tuberculosis deficient in the biosynthesis of some or all forms of p-HBADs was studied. Whilst the mutants grew similarly to the wild-type strain in macrophages and C57BL/6 mice, two of the mutants induced a more severe and diffuse inflammation in the lungs. The lack of production of some or all forms of p-HBADs in these two mutants also correlated with an increased secretion of the pro-inflammatory cytokines tumour-necrosis factor α, interleukin 6 and interleukin 12 in vivo. We propose that the loss of production of p-HBADs by tubercle bacilli results in their diminished ability to suppress the pro-inflammatory response to infection and that this ultimately provokes extensive pulmonary lesions in the C57BL/6 model of tuberculosis infection.

Background

The Mycobacterium tuberculosis genome encodes two peptide transporters encoded by Rv3665c-Rv3662c and Rv1280c-Rv1283c. Both belong to the family of ABC transporters containing two nucleotide-binding subunits, two integral membrane proteins and one substrate-binding polypeptide. However, little is known about their functions in M. tuberculosis. Here we report functional characterization of the Rv1280c-Rv1283c-encoded transporter and its substrate-binding polypeptide OppAMTB.

Methodology/Principal Findings

OppAMTB was capable of binding the tripeptide glutathione and the nonapeptide bradykinin, indicative of a somewhat broad substrate specificity. Amino acid residues G109, N110, N230, D494 and F496, situated at the interface between domains I and III of OppA, were required for optimal peptide binding. Complementaton of an oppA knockout mutant of M. smegmatis with OppAMTB confirmed the role of this transporter in importing glutathione and the importance of the aforesaid amino acid residues in peptide transport. Interestingly, this transporter regulated the ability of M. tuberculosis to lower glutathione levels in infected compared to uninfected macrophages. This ability was partly offset by inactivation of oppD. Concomitantly, inactivation of oppD was associated with lowered levels of methyl glyoxal in infected macrophages and reduced apoptosis-inducing ability of the mutant. The ability to induce the production of the cytokines IL-1β, IL-6 and TNF-α was also compromised after inactivation of oppD.

Conclusions

Taken together, these studies uncover the novel observations that this peptide transporter modulates the innate immune response of macrophages infected with M. tuberculosis.

We report on the identification of a glycosyltransferase (GT) from Mycobacterium tuberculosis H37Rv, Rv3779, of the membranous GT-C superfamily responsible for the direct synthesis of polyprenyl-phospho-mannopyranose and thus indirectly for lipoarabinomannan, lipomannan, and the higher-order phosphatidyl-myo-inositol mannosides.

Objective

To test the safety, tolerability, and pharmacokinetics of the anti- TNF-α monoclonal antibody, infliximab, in subjects with intravenous immunoglobulin (IVIG)-resistant Kawasaki disease (KD).

Study design

We conducted a multicenter, randomized, prospective trial of second IVIG infusion (2 g/kg) versus infliximab (5 mg/kg) in 24 children with acute KD and fever following initial treatment with IVIG. Primary outcome measures were infliximab safety, tolerability, and pharmacokinetics. Secondary outcome measures were duration of fever and changes in markers of inflammation.

Results

Study drug infusions were associated with cessation of fever within 24 hours in 11 of 12 subjects treated with infliximab and 8 of 12 subjects retreated with IVIG. No infusion reactions or serious adverse events were attributed to either study drug. No significant differences were observed between treatment groups in the change from baseline for laboratory variables, fever, or echocardiographic assessment of coronary arteries.

Conclusion

Both infliximab and a second IVIG infusion were safe and well-tolerated in subjects with KD who were resistant to standard IVIG treatment. The optimal management of patients resistant to IVIG remains to be determined.

Nosocomial outbreaks attributable to glutaraldehyde-resistant, rapidly growing mycobacteria are increasing. Here, evidence is provided that defects in porin expression dramatically increase the resistance of Mycobacterium smegmatis and Mycobacterium chelonae to glutaraldehyde and another aldehyde disinfectant, ortho-phthalaldehyde. Since defects in porin activity also dramatically increased the resistance of M. chelonae to drugs, there is thus some concern that the widespread use of glutaraldehyde and ortho-phthalaldehyde in clinical settings may select for drug-resistant bacteria.

Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharide biosynthesis in Mycobacterium spp. Here, the crystallization and preliminary crystallographic analysis of GpgS from M. tuberculosis and of its complex with UDP are reported.

Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharide biosynthesis in mycobacteria. These important molecules are believed to be involved in the regulation of fatty-acid and mycolic acid synthesis. The enzyme belongs to the recently defined GT81 family of retaining glycosyltransferases (CAZy, Carbohydrate-Active Enzymes Database; see http://www.cazy.org). Here, the purification, crystallization and preliminary crystallographic analysis are reported of GpgS from Mycobacterium tuberculosis and of its complex with UDP. GpgS crystals belonged to space group I4, with unit-cell parameters a = 98.85, b = 98.85, c = 127.64 Å, and diffracted to 2.6 Å resolution. GpgS–UDP complex crystals belonged to space group I4, with unit-cell parameters a = 98.32, b = 98.32, c = 127.96 Å, and diffracted to 3.0 Å resolution.

Background

Group A beta hemolytic streptococcus (GABHS) pharyngitis is a common childhood illness. Penicillin remains the gold standard therapy, but macrolides are indicated for the penicillin allergic patient, and are often used for convenience.

Methods

We conducted a surveillance study of children with pharyngitis and positive streptococcal rapid antigen testing from 10/05 to 10/06 at 2 sites (A & B). Demographics, treatment, and resistance data was collected and compared to previous data from 2002. Erythromycin (EM) resistance was determined by disk diffusion and E-test on 500 isolates. Pulse field gel electrophoresis (PFGE) was performed to measure genetic relatedness of isolates. StatXact version 8 software (Cytel Inc., Cambridge, MA) was utilized to perform Fisher's exact test and exact confidence interval (CI) analysis.

Results

There were no differences in resistance rates or demographic features, with the exception of race, between sites A & B. EM resistance was 0 in 2002, 3.5% in 2005-06 at site A, and 4.5% in 2005-06 at site B. 3/7 and 3/9 had inducible resistance at A and B respectively. 8 isolates had relatedness ≥80%, 5 of which were 88% homologous on PFGE.

Conclusion

Community macrolide resistance has increased following increased macrolide use. These results may have treatment implications if use continues to be high.

Synopsis Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharides (MGLP) biosynthesis in Mycobacterium spp. Here we report the crystallization and preliminary crystallographic analysis of GpgS from Mycobacterium tuberculosis and its complex with UDP at 2.6 Å and 3.0 Å resolution, respectively.

Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharides (MGLP) biosynthesis in mycobacteria. These important molecules are believed to be involved in the regulation of fatty acid and mycolic acid synthesis. The enzyme belongs to the recently defined GT81 family of retaining glycosyltransferases (CAZy, Carbohydrate-Active enZymes data base; see www.cazy.org). Here we report the purification, crystallization and preliminary crystallographic analysis of GpgS from Mycobacterium tuberculosis and its complex with UDP. GpgS crystals belong to space group I4, with unit-cell parameters a = 98.85, b = 98.85, c= 127.64 Å, and diffract to 2.6 Å resolution. GpgS-UDP complex crystals belong to space group I4 with unit-cell parameters a= 98.32, b= 98.32, c= 127.96 Å, and diffract to 3.0 Å resolution.

By labeling surface carbohydrates we found that a pool of lipoglycans, cell wall associated, is exposed at the cell surface of mycobacteria and thus, most probably, inserted in the outer leaflet of the outer membrane. In contrast, plasma membrane anchored lipoglycans are not accessible to surface labeling. This result supports the role of lipoglycans as key immunomodulatory molecules but raises the question of their transport from the plasma membrane, where they are synthesized, to the outermost layers of the envelope, where they can act as modulins. The data are discussed in term of consequences for cell envelope organization.

SUMMARY

Mycobacterium tuberculosis and other pathogenic mycobacterial species produce large amounts of a glycogen-like α-glucan that represents the major polysaccharide of their outermost capsular layer. To determine the role of the surface-exposed glucan in the physiology and virulence of these bacteria, orthologs of the glg genes involved in the biosynthesis of glycogen in Escherichia coli were identified in M. tuberculosis H37Rv and inactivated by allelic replacement. Biochemical analyses of the mutants and complemented strains indicated that the synthesis of glucan and glycogen involves the α-1,4-glucosyltransferases Rv3032 and GlgA (Rv1212c), the ADP-glucose pyrophosphorylase GlgC (Rv1213) and the branching enzyme GlgB (Rv1326c). Disruption of glgC reduced by half the glucan and glycogen contents of M. tuberculosis, whereas the inactivation of glgA and Rv3032 affected the production of capsular glucan and glycogen, respectively. Attempts to disrupt Rv3032 in the glgA mutant were unsuccessful, suggesting that a functional copy of at least one of the two α-1,4-glucosyltransferases is required for growth. Importantly, the glgA mutant was impaired in its ability to persist in mice, suggesting a role for the capsular glucan in the persistence phase of infection. Unexpectedly, GlgB was found to be an essential enzyme.

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2′ epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.

Author Summary

Tuberculosis is still a major threat to global health. The disease in humans is caused by a bacterium, Mycobacterium tuberculosis, and treatment of an infected individual requires more than six months of chemotherapy. Because such a long course of treatment is required, compliance is low, which can result in the development of multidrug resistant strains (MDR-TB) and even extremely resistant strains (XDR-TB). Identifying new drug targets and potential lead therapeutic compounds are needed to combat MDR-XDR-TB. We developed a new type of assay based on the visualization of mycobacterium replication within host cells and applied it for the search of compounds that are able to chase the pathogen from its hideout. As a result, we found 20 new series of drug candidates that are effective against the bacilli in its hiding place, potentially addressing a crucial aspect in the resilience of the disease. We also showed that one series of compounds acts by inhibiting a key enzyme required for the synthesis of an essential component from the mycobacterial cell wall that is not targeted by any of the commercially available antituberculosis drugs. Altogether, our results pave the way for development of the next generation of antibacterial agents.

Background

Mycobacteria produce two unique families of cytoplasmic polymethylated polysaccharides - the methylglucose lipopolysaccharides (MGLPs) and the methylmannose polysaccharides (MMPs) - the physiological functions of which are still poorly defined. Towards defining the roles of these polysaccharides in mycobacterial physiology, we generated knock-out mutations of genes in their putative biosynthetic pathways.

Methodology/Principal Findings

We report here on the characterization of the Rv1208 protein of Mycobacterium tuberculosis and its ortholog in Mycobacterium smegmatis (MSMEG_5084) as the enzymes responsible for the transfer of the first glucose residue of MGLPs. Disruption of MSMEG_5084 in M. smegmatis resulted in a dramatic decrease in MGLP synthesis directly attributable to the almost complete abolition of glucosyl-3-phosphoglycerate synthase activity in this strain. Synthesis of MGLPs in the mutant was restored upon complementation with wild-type copies of the Rv1208 gene from M. tuberculosis or MSMEG_5084 from M. smegmatis.

Conclusions/Significance

This is the first evidence linking Rv1208 to MGLP biosynthesis. Thus, the first step in the initiation of MGLP biosynthesis in mycobacteria has been defined, and subsequent steps can be inferred.

Similarities between Mycobacterium tuberculosis phoP-phoR mutants and the attenuated laboratory strain M. tuberculosis H37Ra in terms of morphological and cytochemical properties, lipid content, gene expression and virulence attenuation prompted us to analyze the functionality of this two-component regulator in the latter strain. Sequence analysis revealed a base substitution resulting in a one-amino-acid change in the likely DNA-binding region of PhoP in H37Ra relative to H37Rv. Using gel-shift assays, we show that this mutation abrogates the ability of the H37Ra PhoP protein to bind to a 40-bp segment of its own promoter. Consistent with this result, the phoP gene from H37Rv but not that from H37Ra was able to restore the synthesis of sulfolipids, diacyltrehaloses and polyacyltrehaloses in an isogenic phoP-phoR knock-out mutant of M. tuberculosis Moreover, complementation of H37Ra with phoP from H37Rv fully restored sulfolipid, diacyltrehalose and polyacyltrehalose synthesis, clearly indicating that the lack of production of these lipids in H37Ra is solely due to the point mutation in phoP. Using a pks2-3/4 knock-out mutant of M. tuberculosis H37Rv, evidence is further provided that the above-mentioned polyketide-derived acyltrehaloses do not significantly contribute to the virulence of the tubercle bacillus in a mouse model of infection. Reasons for the attenuation of H37Ra thus most likely stand in other virulence factors, many of which are expected to belong to the PhoP regulon and another of which, unrelated to PhoP, appears to be the lack of production of phthiocerol dimycocerosates in this strain.